July 1, 2008 (Vol. 28, No. 13)

Unigene Has Introduced an Enzyme-Based Assay for the Discovery of Amidated Peptides

Peptides are among the most biologically relevant molecules known and also among the most productive areas of pharmaceutical R&D. According to Frost and Sullivan, more than 40 peptide drugs are on the market with close to 300 more in clinical testing.

Approved agents include the natural peptides such as insulin, oxytocin, exendin-4, parathyroid hormone, and calcitonin. Several synthetic or derivatized peptides or peptide analogs have also been developed into successful products such as Fuzeon®, Integrilin®, DDAVP®, Sandostatin®, Lupron®, and Symlin®. One form of insulin alone, Lilly’s Humalog®, enjoyed sales of $1.1 billion in 2004.

What does it take to introduce a new peptide drug? In addition to discovery, development, and success in the clinic, developers need to address the problem of scale up in manufacturing and eventually drug delivery.

Most of the currently approved peptide drugs including insulin are administered by injection or infusion. At one time, the well-known drawbacks of injectable dosage forms held back the development of peptide drugs. That is no longer the case.

The pipeline of nearly every pharmaceutical company today includes peptides or peptide-like drugs. Some firms like Novo Nordisk (www.novonordisk.com) have shifted their discovery and development efforts almost entirely to peptides.

A Cornucopia for Discovery

Peptides or proteins regulate most human physiology through the endocrine/paracrine systems and serve as hormones, neurotransmitters, growth factors, enzymes, and also as structural components of cells.

Insulin was the first peptide introduced into human clinical practice. Discovered by Banting in 1921, this 51-amino acid peptide with a molecular weight of 5808 daltons quickly revolutionized the treatment of diabetes.

Despite the introduction of numerous peptide drugs since the 1920s, insulin remains the leading peptide therapeutic and a perennial blockbuster both in its native and chemically modified forms.

One strategy for discovering new peptide therapies involves chemical modification of known active peptides.

For example, esterification, oligomerization, or amino-acid substitution can in some cases enhance a peptide’s activity while conferring on it desirable pharmacokinetic properties such as extended plasma half-life.

Insulin has been introduced in several formats and delivery vehicles including buccal, rectal, sub-lingual, and more recently, inhaled and intranasal dosage forms. Diabetics now use both slow-acting insulin products to maintain basal levels of the drug and fast-acting forms taken at mealtime.

Although many pharmacologically relevant peptides are already known, the human genome project is expected to reveal dozens, if not hundreds, of additional peptides with regulatory and disease-modulating activity. We expect that within a few years the discovery of naturally occurring peptides will rival in importance the recent discovery of several classes of small regulatory RNAs.

The analogy here is striking, since as recently as 2000 no one even knew small regulatory RNAs existed, much less understood their far-reaching influence on human health.

Discovering new peptide drugs will require significant advances in how researchers identify, quantify, and analyze peptides as small as three amino acids in length.

Considering the number and concentrations of confounding molecules in biological samples, that task will not be easy. Given the huge dynamic range for biological molecules, traditional analytical methods such as mass spectrometry and HPLC-MS will require highly selective and innovative sample-preparation methods to identify peptides whose existence in blood may be only fleeting.

Unigene Laboratories (www.unigene.com) has developed a new enzyme-based assay for the discovery of amidated peptides that ultimately should identify low-concentration peptide hormones and will complement LC and LC/MS methods, thereby fueling the discovery of many more biologically active peptides suitable for pharmaceutical development.

Delivery Is a Crucial Component

Orally delivered peptides and proteins face a hostile gastric environment, proteolytic enzymes in the stomach and intestine, and the intestinal permeability barrier. These obstacles result in relatively low bioavailability of orally delivered formulations, which, in turn, places a burden on manufacturing of peptides due to the large doses needed.

Peptides’ large size and hydrophilicity severely limit their absorption through the GI tract. Peptides are also susceptible to degradation by enzymes in the stomach, primarily pepsin, and the stomach’s acidic environment.

Peptides surviving passage through the stomach are susceptible to cleavage by intestinal proteases secreted from the pancreas or localized on the brush border membranes of intestinal epithelia.

The mucus layer of the GI tract, which binds polar molecules, serves as a further barrier to absorption through the lumen of the intestine.

Consequently, the bioavailability of peptides of more than two to three amino acids is extremely poor.

The rational design of orally active peptide pharmaceuticals should therefore strive to inhibit or modulate proteolysis, enhance absorption in the stomach or intestine by facilitating paracellular or transcellular transport and/or by increasing penetration through the mucus barrier, and increase the circulating half-life of the peptide in situations requiring sustained presence for therapeutic efficacy.

Several technologies fulfilling some or all of these goals have been tested in animals and humans. Unigene’s oral delivery technology, which requires no chemical modification of the peptide, uses up to four groups of excipients, depending on the peptide to be delivered. Excipient groups are composed of organic acids that serve as general protease inhibitors or modulators, enhancers of paracellular transport, detergents for improving peptide solubility and transport while reducing interaction with mucus, and protease-specific inhibitors for enhancing circulating half-life.

The enteric coating confers stability to a capsule or tablet to acidic pH, allowing it to pass through the stomach intact. As the pH in the intestine increases above 5.5, the coating dissolves and releases peptide and excipients into a localized area of the intestine.

Unigene has prepared capsules and tablets and obtained human pharmacokinetic data that demonstrates absorption of intact peptide into the systemic circulation. For example, the oral delivery of salmon calcitonin, an amidated 32-amino acid peptide for treating postmenopausal osteoporosis and hypercalcemia of malignancy, is shown in the Figure.

Unigene has also demonstrated delivery in animals for luteinizing hormone-releasing hormone (LHRH), leuprolide, desmopressin, PTH analogs, insulin, glucogen-like peptide-1 and other glucose regulatory peptides. It has been determined that bioavailability depends not only on the size and charge of the peptide, but also on the presence of structural features that render the peptide more protease resistant such as blocked N- and C-termini or the incorporation of D-amino acids.


The low bioavailability of orally delivered peptides, which ranges from 1% to 10%, should not be problematic from a therapeutic standpoint since most peptides are highly potent. However, it carries special significance for manufacturing cost and scale, particularly for peptide drugs with high dosing requirements (e.g., Fuzeon and insulin).

Although great strides have been made in reducing costs and improving scalability for peptide production through chemical synthesis, recombinant technology has become the method of choice for the large-scale manufacture of larger peptides (25 amino acids or more).

Recombinant expression of foreign proteins in microorganisms and cells provides the best combination of cost-effectiveness, scalability, and environmental safety.

The manufacture of peptides in recombinant organisms began in the early 1980s. Since then, many different host cells and organism types have been used to produce peptides.

Microbial fermentation, particularly in E. coli, has several significant advantages over mammalian cell culture. E. coli fermentations are rapid, predictable, free of downstream contaminants associated with cells, and less costly than cell culture.

Conventional bacterial fermentation systems are not without their limitations, however. For example, the relatively small size and lack of tertiary structure of most peptides makes them susceptible to rapid degradation in the cytoplasm of expressing bacteria and yeast.

This drawback may be mitigated by expressing the product with a much larger protein fusion partner, which generally protects the peptide from proteolysis. Liberation of the product from the fusion partner, however, requires chemical or enzymatic cleavage, which adds at least two processing steps (cleavage and purification) and results in significantly reduced peptide yield.

Also, lysing the bacterial cell to release the peptide product causes release of all the bacterial proteins as well as DNA and bacterial endotoxins. These process-related contaminants then need to be purified away from the peptide of interest, which further increases the number of purification steps required.

An ideal expression system, therefore, would be one that allowed for the production of peptides without a fusion partner and secreted the expressed peptide from the cell into the growth medium, thus leaving the bacterial cells intact.

Unfortunately, obtaining excreted products from E. coli is difficult because the organisms do not normally excrete peptide or protein products and they produce proteases that break down foreign proteins intracellularly.

A further complication in producing peptide hormones in bacteria or yeast is the frequent requirement that these products be amidated at the C-terminus of the hormone for full biological activity. Prokaryotes lack peptidylglycine a-amidating monooxygenase (PAM), the enzyme that carries out this post-translational amidation, therefore, peptides produced in E. coli are not C-terminally amidated.

To address these issues, Unigene has developed a manufacturing platform that efficiently produces amidated peptide hormones through the use of two recombinant cell lines. The glycine-extended precursor of the desired peptide is first produced in recombinant E. coli using a direct expression technology.

The expression construct incorporates an upstream signal sequence that causes the peptide to translocate from the cytoplasm to the periplasm, at which point the signal sequence is cleaved. Due to further innovations in the growth conditions and the components of the growth medium, the peptide is then excreted into the growth medium. The E. coli host cell is a protease-minus cell that allows for the accumulation of the peptide in the growth medium without significant degradation.

Since E. coli does not excrete appreciable quantities of endogenous proteins, the peptide product in the conditioned medium provides a relatively enriched starting material for purification, thus reducing the number of purification steps and increasing the yields from purification.

After purification, the peptide is treated in vitro with PAM, which is separately produced from recombinant CHO cells. PAM quantitatively converts a variety of C-terminally glycine-extended peptides to the corresponding peptide amides at a mass ratio of enzyme to substrate of 1:1000 or greater, depending on the glycine residue’s immediate neighbor. Hence, the quantity of PAM needed is a small fraction of the amount of peptide to be produced, and the higher cost of production of PAM in CHO cells does not add appreciably to the overall cost of the process.

One or more chromatography steps then separates amidated product from precursor and other minor contaminants. After purification and amidation, the peptide is typically >98% pure.

The direct expression process is readily scalable up to 20,000 liters with no loss of productivity. Yields will vary depending on the peptide, but such products as salmon calcitonin, parathyroid hormone analogs, glucose regulatory peptide analogs, secretin, and growth hormone releasing factor have been expressed at up to 1g/liter of intact peptide. In instances where peptide degradation occurred in the growth medium, changes in the nutrient feed significantly reduced it.

There has never been a more exciting time to be involved in peptide pharmaceutical development. Oral delivery methods have changed the paradigm for peptide drugs irrevocably and for the better. The results of human genome research should provide peptide drug candidates for years to come.

Warren P. Levy, Ph.D., is CEO and president of Unigene Laboratories. Web: www.unigene.com.Email: [email protected].

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